Project Summary Regulated production of reactive oxygen and nitrogen species serves important roles in our biology, such as providing unique mechanisms for cell signaling, as well as defense against invading pathogens. However, their elevated levels are associated with numerous human diseases including atherosclerosis, stroke, neurodegeneration, inflammation, etc. These reactive agents modify redox-active amino acid residues on proteins, causing structural and functional changes that serve as the molecular basis for signaling, as well as the pathogenesis of numerous diseases. Several oxidative and nitrative post- translational modifications (PTMs) of tryptophan have been recently identified in many sites of our proteome. These modifications are not distributed randomly; rather, specific tryptophan residues on specific proteins are found to be selectively modified. While a limited number of reports have shown that these modifications can trigger alterations in protein structure and function, physiological consequences of the tryptophan modifications observed in our proteome remains mostly unclear. At the core of this knowledge gap lies our current inability to generate target proteins in a homogeneously modified form, and ask how their properties are altered in vivo and in vitro. To overcome this limitation, here we propose the development of technology that will enable co-translational site-specific incorporation of physiologically relevant modified tryptophan residues into any target protein. We have recently developed a unique tryptophanyl-tRNA synthetase (TrpRS)/tRNATrp pair that can be used to site-specifically incorporate unnatural amino acids into proteins expressed in both E. coli and eukaryotic cells. We have further demonstrated our ability to engineer this TrpRS/tRNATrp pair to enable site-specific incorporation of a variety of tryptophan analogs into proteins. Here we propose further development of this platform to allow co-translational site-specific incorporation of physiologically relevant oxidized/nitrated tryptophan derivatives, which will for the first time enable facile expression of target proteins harboring these modified tryptophans at predefined sites in both E. coli as well as mammalian cells. We will further use this platform to investigate the role of tryptophan nitration using two established targets, phosphoglycerate kinase 1 and ?-enolase, both important human metabolic proteins. Our work will establish a novel and general approach for understanding the role of oxidative/nitrative modifications of tryptophan residues in human health and disease. The ability to characterize the elusive connections between oxidative/nitrative tryptophan modification and various human diseases will also uncover new opportunities for therapeutic intervention.